Histone Demethylase Research Articles

Empowering γδ T-cell functionality with vitamin C.

Vitamin C (ascorbic acid) is a potent antioxidant and a cofactor for various enzymes including histone demethylases and methylcytosine dioxygenases. Vitamin C also exerts direct cytotoxicity toward selected tumor cells including colorectal carcinoma. Moreover, vitamin C has been shown to impact immune cell differentiation at various levels including maturation and/or functionality of T cells and their progenitors, dendritic cells, B cells, and NK cells. γδ T cells have recently attracted great interest as effector cells for cell-based cancer immunotherapy, due to their HLA-independent recognition of a large variety of tumor cells. While γδ T cells can thus be also applied as an allogeneic off-the-shelf product, it is obvious that the effector function of γδ T cells needs to be optimized to ensure the best possible clinical efficacy. Here we review the immunomodulatory mechanisms of vitamin C with a special focus on how vitamin C enhances the effector function of γδ T cells. We also discuss future directions of how vitamin C can be used in the clinical setting to boost the efficacy of adoptive cell therapies.

Transcription factor Olig2 is a major downstream effector of histone demethylase Phf8 during oligodendroglial development.

The plant homeodomain finger protein Phf8 is a histone demethylase implicated by mutation in mice and humans in neural crest defects and neurodevelopmental disturbances. Considering its widespread expression in cell types of the central nervous system, we set out to determine the role of Phf8 in oligodendroglial cells to clarify whether oligodendroglial defects are a possible contributing factor to Phf8-dependent neurodevelopmental disorders. Using loss- and gain-of-function approaches in oligodendroglial cell lines and primary cell cultures, we show that Phf8 promotes the proliferation of rodent oligodendrocyte progenitor cells and impairs their differentiation to oligodendrocytes. Intriguingly, Phf8 has a strong positive impact on Olig2 expression by acting on several regulatory regions of the gene and changing their histone modification profile. Taking the influence of Olig2 levels on oligodendroglial proliferation and differentiation into account, Olig2 likely acts as an important downstream effector of Phf8 in these cells. In line with such an effector function, ectopic Olig2 expression in Phf8-deficient cells rescues the proliferation defect. Additionally, generation of human oligodendrocytes from induced pluripotent stem cells did not require PHF8 in a system that relies on forced expression of Olig2 during oligodendroglial induction. We conclude that Phf8 may impact nervous system development at least in part through its action in oligodendroglial cells.

Identification of KDM4C as a gene conferring drug resistance in multiple myeloma.

Bortezomib (BTZ), a proteasome inhibitor, is a promising therapeutic option for multiple myeloma (MM) patients. However, drug resistance often occurs, leading to disease relapse and poor prognosis. In this study, we aimed to identify novel genes associated with drug resistance and investigate their roles in BTZ resistance. Through the screening of 26 genes frequently associated with chemosensitivity or drug resistance, we discovered that KDM4C, a histone demethylase, exhibited increased expression in BTZ-resistant MM cells compared to their sensitive counterparts. Overexpression of KDM4C enhanced the tolerance of a MM cell line to the drug, whereas the knockdown of KDM4C, using shRNA, increased the sensitivity of resistant cells to BTZ treatment. This suggests that KDM4C plays a pivotal role in conferring BTZ resistance. Our study offers fresh insights into BTZ resistance in MM and highlights KDM4C as a potential target for overcoming drug resistance.

Structural simulation and selective inhibitor discovery study for histone demethylases KDM4E/6B from a computational perspective

The methylation and demethylation of lysine and arginine side chains are fundamental processes in gene regulation and disease development. Histone lysine methylation, controlled by histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs), plays a vital role in maintaining cellular homeostasis and has been implicated in diseases such as cancer and aging. This study focuses on two members of the lysine demethylase (KDM) family, KDM4E and KDM6B, which are significant in gene regulation and disease pathogenesis. KDM4E demonstrates selectivity for gene regulation, particularly concerning cancer, while KDM6B is implicated in inflammation and cancer. The study utilizes specific inhibitors, DA-24905 and GSK-J1, showcasing their exceptional selectivity for KDM4E and KDM6B, respectively. Employing an array of computational simulations, including sequence alignment, molecular docking, dynamics simulations, and free energy calculations, we conclude that although the binding cavities of KDM4E and KDM6B has high similarity, there are still some different crucial amino acid residues, indicating diverse binding forms between protein and ligands. Various interaction predominates when proteins are bound to different ligands, which also has significant effect on selective inhibition. These findings provide insights into potential therapeutic strategies for diseases by selectively targeting these KDM members.

Potential role of lipophagy impairment for anticancer effects of glycolysis-suppressed pancreatic ductal adenocarcinoma cells

Although increased aerobic glycolysis is common in various cancers, pancreatic ductal adenocarcinoma (PDAC) cells can survive a state of glycolysis suppression. We aimed to identify potential therapeutic targets in glycolysis-suppressed PDAC cells. By screening anticancer metabolic compounds, we identified SP-2509, an inhibitor of lysine-specific histone demethylase 1A (LSD1), which dramatically decreased the growth of PDAC PANC-1 cells and showed an anti-tumoral effect in tumor-bearing mice. The growth of glycolysis-suppressed PANC-1 cells was also inhibited by another LSD1 inhibitor, OG-L002. Similarly, the other two PDAC cells (PK-1 and KLM-1) with suppressed glycolysis exhibited anticancer effects against SP-2509. However, the anticancer effects on PDAC cells were unrelated to LSD1. To investigate how PDAC cells survive in a glycolysis-suppressed condition, we conducted proteomic analyses. These results combined with our previous findings suggested that glucose-starvation causes PDAC cells to enhance mitochondrial oxidative phosphorylation. In particular, mitochondrial fatty acid metabolism was identified as a key factor contributing to the survival of PDAC cells under glycolysis suppression. We further demonstrated that SP-2509 and OG-L002 disturbed fatty acid metabolism and induced lipid droplet accumulation through the impairment of lipophagy, but not bulk autophagy. These findings indicate a significant potential association of lipophagy and anticancer effects in glycolysis-suppressed PDAC cells, offering ideas for new therapeutic strategies for PDAC by dual inhibition of glycolysis and fatty acids metabolism.

Targeting histone demethylases JMJD3 and UTX: selenium as a potential therapeutic agent for cervical cancer

BackgroundThe intriguing connection between selenium and cancer resembles a captivating puzzle that keeps researchers engaged and curious. While selenium has shown promise in reducing cancer risks through supplementation, its interaction with epigenetics in cervical cancer remains a fascinating yet largely unexplored realm. Unraveling the intricacies of selenium's role and its interaction with epigenetic factors could unlock valuable insights in the battle against this complex disease.ResultSelenium has shown remarkable inhibitory effects on cervical cancer cells in various ways. In in vitro studies, it effectively inhibits the proliferation, migration, and invasion of cervical cancer cells, while promoting apoptosis. Selenium also demonstrates significant inhibitory effects on human cervical cancer-derived organoids. Furthermore, in an in vivo study, the administration of selenium dioxide solution effectively suppresses the growth of cervical cancer tumors in mice. One of the mechanisms behind selenium's inhibitory effects is its ability to inhibit histone demethylases, specifically JMJD3 and UTX. This inhibition is observed both in vitro and in vivo. Notably, when JMJD3 and UTX are inhibited with GSK-J4, similar biological effects are observed in both in vitro and in vivo models, effectively inhibiting organoid models derived from cervical cancer patients. Inhibiting JMJD3 and UTX also induces G2/M phase arrest, promotes cellular apoptosis, and reverses epithelial-mesenchymal transition (EMT). ChIP-qPCR analysis confirms that JMJD3 and UTX inhibition increases the recruitment of a specific histone modification, H3K27me3, to the transcription start sites (TSS) of target genes in cervical cancer cells (HeLa and SiHa cells). Furthermore, the expressions of JMJD3 and UTX are found to be significantly higher in cervical cancer tissues compared to adjacent normal cervical tissues, suggesting their potential as therapeutic targets.ConclusionsOur study highlights the significant inhibitory effects of selenium on the growth, migration, and invasion of cervical cancer cells, promoting apoptosis and displaying promising potential as a therapeutic agent. We identified the histone demethylases JMJD3 and UTX as specific targets of selenium, and their inhibition replicates the observed effects on cancer cell behavior. These findings suggest that JMJD3 and UTX could be valuable targets for selenium-based treatments of cervical cancer.

DNA methylation remodeling and the functional implication during male gametogenesis in rice

BackgroundEpigenetic marks are reprogrammed during sexual reproduction. In flowering plants, DNA methylation is only partially remodeled in the gametes and the zygote. However, the timing and functional significance of the remodeling during plant gametogenesis remain obscure.ResultsHere we show that DNA methylation remodeling starts after male meiosis in rice, with non-CG methylation, particularly at CHG sites, being first enhanced in the microspore and subsequently decreased in sperm. Functional analysis of rice CHG methyltransferase genes CMT3a and CMT3b indicates that CMT3a functions as the major CHG methyltransferase in rice meiocyte, while CMT3b is responsible for the increase of CHG methylation in microspore. The function of the two histone demethylases JMJ706 and JMJ707 that remove H3K9me2 may contribute to the decreased CHG methylation in sperm. During male gametogenesis CMT3a mainly silences TE and TE-related genes while CMT3b is required for repression of genes encoding factors involved in transcriptional and translational activities. In addition, CMT3b functions to repress zygotic gene expression in egg and participates in establishing the zygotic epigenome upon fertilization.ConclusionCollectively, the results indicate that DNA methylation is dynamically remodeled during male gametogenesis, distinguish the function of CMT3a and CMT3b in sex cells, and underpin the functional significance of DNA methylation remodeling during rice reproduction.

Glutamine-mediated epigenetic regulation of cFLIP underlies resistance to TRAIL in pancreatic cancer

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it kills cancer cells while sparing normal cells. However, many cancers, including pancreatic ductal adenocarcinoma (PDAC), exhibit intrinsic or acquired resistance to TRAIL, and the molecular mechanisms underlying TRAIL resistance in cancers, particularly in PDAC, remain unclear. In this study, we demonstrated that glutamine (Gln) endows PDAC cells with resistance to TRAIL through KDM4C-mediated epigenetic regulation of cFLIP. Inhibition of glutaminolysis significantly reduced the cFLIP level, leading to TRAIL-mediated formation of death-inducing signaling complexes. Overexpression of cFLIP dramatically rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Alpha-Ketoglutarate (aKG) supplementation significantly reversed the decrease in the cFLIP level induced by glutaminolysis inhibition and rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Knockdown of glutamic-oxaloacetic transaminase 2, which facilitates the conversion of oxaloacetate and glutamate into aspartate and aKG, decreased aKG production and the cFLIP level and activated TRAIL-induced apoptosis. AKG-mediated epigenetic regulation was necessary for maintaining a high level of cFLIP. Glutaminolysis inhibition increased the abundance of H3K9me3 in the cFLIP promoter, indicating that Gln-derived aKG production is important for Jumonji-domain histone demethylase (JHDM)-mediated cFLIP regulation. The JHDM KDM4C regulated cFLIP expression by binding to its promoter, and KDM4C knockdown sensitized PDAC cells to TRAIL-induced apoptosis. The present findings suggest that Gln-derived aKG production is required for KDM4C-mediated epigenetic regulation of cFLIP, which leads to resistance to TRAIL.

JMJD3 activation contributes to renal protection and regeneration following acute kidney injury in mice.

We have recently demonstrated that Jumonji domain-containing protein D3 (JMJD3), a histone demethylase of histone H3 on lysine 27 (H3K27me3), is protective against renal fibrosis, but its role in acute kidney injury (AKI) remains unexplored. Here, we report that JMJD3 activity is required for renal protection and regeneration in murine models of AKI induced by ischemia/reperfusion (I/R) and folic acid (FA). Injury to the kidney upregulated JMJD3 expression and induced expression of H3K27me3, which was coincident with renal dysfunction, renal tubular cell injury/apoptosis, and proliferation. Blocking JMJD3 activity by GSKJ4 led to worsening renal dysfunction and pathological changes by aggravating tubular epithelial cell injury and apoptosis in both murine models of AKI. JMJD3 inhibition by GSKJ4 also reduced renal tubular cell proliferation and suppressed expression of cyclin E and phosphorylation of CDK2, but increased p21 expression in the injured kidney. Furthermore, inactivation of JMJD3 enhanced I/R- or FA-induced expression of TGF-β1, vimentin, and Snail, phosphorylation of Smad3, STAT3, and NF-κB, and increased renal infiltration by F4/80 (+) macrophages. Finally, GSKJ4 treatment caused further downregulation of Klotho, BMP-7, Smad7, and E-cadherin, all of which are associated with renal protection and have anti-fibrotic effects. Therefore, these data provide strong evidence that JMJD3 activation contributes to renal tubular epithelial cell survival and regeneration after AKI.

Complex formation of ML324, the histone demethylase inhibitor, with essential metal ions: Relationship between solution chemistry and anticancer activity

N-(3-(dimethylamino)propyl-4-(8-hydroxyquinolin-6-yl)benzamide (ML324, HL) is a potent inhibitor of the iron-containing histone demethylase KDM4, a recognized potential target of cancer therapeutics. Herein, we report the proton dissociation and complex formation processes of ML324 with essential metal ions such as Fe(II), Fe(III), Cu(II) and Zn(II) using UV–visible, fluorescence, electron paramagnetic resonance and 1H NMR spectroscopic methods. The electrochemical behaviour of the copper and iron complexes was characterized by cyclic voltammetry and spectroelectrochemistry. The solid phase structure of ML324 analysed by X-ray crystallography is also provided. Based on the solution equilibrium data, ML324 is present in solution in H2L+ form with a protonated dimethylammonium moiety at pH 7.4, and this (N,O) donor bearing ligand forms mono and bis complexes with all the studied metal ions and the tris-ligand species is also observed with Fe(III). At pH 7.4 the metal binding ability of ML324 follows the order: Fe(II) < Zn(II) < Cu(II) < Fe(III). Complexation with iron resulted in a negative redox potential (E'1/2 = −145 mV vs. NHE), further suggesting that the ligand has a preference for Fe(III) over Fe(II). ML324 was tested for its anticancer activity in chemosensitive and resistant human cancer cells overexpressing the efflux pump P-glycoprotein. ML324 exerted similar activity in all tested cells (IC50 = 1.9–3.6 μM). Co-incubation and complexation of the compound with Cu(II) and Zn(II) had no impact on the cytotoxicity of ML324, whereas Fe(III) decreased the toxicity in a concentration-dependent manner, and this effect was more pronounced in the multidrug resistant cells.

Transcriptomic dysregulation and autistic-like behaviors in Kmt2c haploinsufficient mice rescued by an LSD1 inhibitor.

Recent studies have consistently demonstrated that the regulation of chromatin and gene transcription plays a pivotal role in the pathogenesis of neurodevelopmental disorders. Among many genes involved in these pathways, KMT2C, encoding one of the six known histone H3 lysine 4 (H3K4) methyltransferases in humans and rodents, was identified as a gene whose heterozygous loss-of-function variants are causally associated with autism spectrum disorder (ASD) and the Kleefstra syndrome phenotypic spectrum. However, little is known about how KMT2C haploinsufficiency causes neurodevelopmental deficits and how these conditions can be treated. To address this, we developed and analyzed genetically engineered mice with a heterozygous frameshift mutation of Kmt2c (Kmt2c+/fs mice) as a disease model with high etiological validity. In a series of behavioral analyses, the mutant mice exhibit autistic-like behaviors such as impairments in sociality, flexibility, and working memory, demonstrating their face validity as an ASD model. To investigate the molecular basis of the observed abnormalities, we performed a transcriptomic analysis of their bulk adult brains and found that ASD risk genes were specifically enriched in the upregulated differentially expressed genes (DEGs), whereas KMT2C peaks detected by ChIP-seq were significantly co-localized with the downregulated genes, suggesting an important role of putative indirect effects of Kmt2c haploinsufficiency. We further performed single-cell RNA sequencing of newborn mouse brains to obtain cell type-resolved insights at an earlier stage. By integrating findings from ASD exome sequencing, genome-wide association, and postmortem brain studies to characterize DEGs in each cell cluster, we found strong ASD-associated transcriptomic changes in radial glia and immature neurons with no obvious bias toward upregulated or downregulated DEGs. On the other hand, there was no significant gross change in the cellular composition. Lastly, we explored potential therapeutic agents and demonstrate that vafidemstat, a lysine-specific histone demethylase 1 (LSD1) inhibitor that was effective in other models of neuropsychiatric/neurodevelopmental disorders, ameliorates impairments in sociality but not working memory in adult Kmt2c+/fs mice. Intriguingly, the administration of vafidemstat was shown to alter the vast majority of DEGs in the direction to normalize the transcriptomic abnormalities in the mutant mice (94.3 and 82.5% of the significant upregulated and downregulated DEGs, respectively, P < 2.2 × 10-16, binomial test), which could be the molecular mechanism underlying the behavioral rescuing. In summary, our study expands the repertoire of ASD models with high etiological and face validity, elucidates the cell-type resolved molecular alterations due to Kmt2c haploinsufficiency, and demonstrates the efficacy of an LSD1 inhibitor that might be generalizable to multiple categories of psychiatric disorders along with a better understanding of its presumed mechanisms of action.

MLL1 histone methyltransferase and UTX histone demethylase functionally cooperate to regulate the expression of NRF2 in response to ROS-induced oxidative stress

The transcription factor NRF2 plays a pivotal role in maintaining redox and metabolic homeostasis by orchestrating oxidative stress-dependent transcription programs. Despite growing evidence implicating various cellular components in the regulation of NRF2 activity at the posttranslational stage, relatively less is known about the factors dictating the transcriptional activation of NRF2 in response to oxidative stress. In this study, we report the crucial roles of MLL1, an H3K4-specific methyltransferase, and UTX, an H3K27-specific histone demethylase, in the NRF2-dependent transcription program under oxidative stress. We find that the depletion of MLL1 or UTX results in increased susceptibility to oxidative stress, accompanied by higher intracellular ROS and the failed activation of antioxidant genes, including NRF2. In addition, MLL1 and UTX selectively target the NRF2 promoter, and exogenous FLAG-NRF2 expression increases the viability of MLL1-or UTX-depleted cells upon exposure to hydrogen peroxide. RNA-seq analysis demonstrates that depletion of MLL1 or UTX affects the changes in NRF2-dependent transcriptome in response to oxidative stress. Furthermore, ChIP and ChIP-seq analyses find that MLL1 and UTX functionally cooperate to establish a chromatin environment that favors active transcription at the H3K4me3/H3K27me3 bivalent NRF2 promoter in response to ROS-induced oxidative stress. Collectively, these findings provide a molecular mechanism underlying the cellular response to oxidative stress and highlight the importance of the chromatin structure and function in maintaining redox homeostasis.

Abstract 307: Generation and characterization of a glioblastoma stem cell model with acquired resistance to LSD1 inhibition

Abstract Epigenetic modifications play an important role in the initiation of various cancer types, including glioblastoma (GBM), and in the development of therapeutic resistance. Patients with GBM, a common malignant brain tumor, have an average survival of 12-15 months after first-line therapy. Lysine-specific demethylase 1 (LSD1) is a histone demethylase that is overexpressed in glioblastoma stem cells (GSCs) and functions to promote proliferation, inhibit differentiation, and enhance motility. Previous work from our group has investigated LSD1 as a therapeutic target in glioblastoma and identified genes that may predict resistance to pharmacological LSD1 inhibitors using RNA-seq. Five upregulated genes, HKDC1, RAB3IL1, RAB39B, FTH1, and FAM213A, were associated with LSD1 inhibitor (LSD1i) resistance and were validated in brain tissue of a GBM model treated with GSK-LSD1 after succumbing to tumor burden. In order to determine whether selection pressure from LSD1 inhibitor treatment caused upregulation of these genes, we created a model of acquired resistance in GSC lines in vitro. Isogenic resistant lines were generated by treating MDA-GSC17 cells with increasing concentrations of LSD1i or diluent (DMSO), as tolerated starting at 50 uM (which was five fold below the IC50 for these lines). In parallel, MDA-GSC17 controls were exposed to DMSO. The resistant line was created over 4 weeks and monitored by cell number and viability to reach 90%. To assess the change in gene expression, we conducted quantitative PCR for the five genes in the parental and resistant MDA-GSC17 line and saw upregulation in 3 of the 5 genes. Two genes, HKDC1 and RAB3IL1, were unable to be assessed due to low basal expression. However, 3 genes were upregulated which are RAB39B, FTH1, and FAM213A. RAB39B is a Ras-analog in brain (RAB) small GTPase and a member of the RAS oncogene family, involved in vesicular trafficking. High expression of ferritin heavy chain 1 (FTH1) is correlated with high-grade gliomas and poor prognosis in glioblastoma. Lastly, the redox regulatory protein, FAM213A, can activate antioxidant proteins and elevated expression is associated with worse outcomes for AML patients. This suggests that modulating the RAS signaling pathways and specific redox pathways may overcome LSD1 resistance. Understanding the changes in gene expression as the cells acquire resistance to LSD1 inhibitors will help us understand the mechanism of resistance to LSD1 inhibitors in glioblastoma and to design new treatment strategies. Citation Format: Kareena H. Patel, Lea Stitzlein, Deokhwa Nam, Frederick Lang, Joy Gumin, Joya Chandra. Generation and characterization of a glioblastoma stem cell model with acquired resistance to LSD1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 307.

Abstract 3346: JMJD6 as a novel tumorigenic factor and therapeutic target in group 3 (MYC-driven) medulloblastoma

Abstract Medulloblastoma (MB) is the most common type of childhood brain cancer worldwide. Although current treatment with surgery and extensive chemoradiation has led to increased survival rates, many MB patients still die from the disease. Moreover, surviving patients suffer severe long-term side effects as a consequence of treatment. It is therefore crucial to develop more effective and less toxic therapies. The most aggressive subtype of MB tumors often exhibits amplification or overexpression of the MYC oncogene. Patients with MYC-amplified MB exhibit a high frequency of cerebrospinal tumor dissemination, often experience treatment resistance and have extremely poor prognoses. While the MYC oncogene is established as the oncogenic driver in Group 3 MB, it has remained undruggable. Thus, targeting regulatory components of MYC and the signaling pathways regulated by it is of great potential therapeutic value. Studies have revealed that MB has very few germline mutations in cancer predisposition genes, suggesting that dysregulated epigenetic pathways might be critical in MB pathogenesis. Particularly, dysregulation of epigenetic modifiers, including histone methyltransferases and histone demethylases, is very common in Group 3 MB, compared to other MB subgroups. Therefore, it is important to identify such epigenetic modifiers that may have controls on MYC and its tumorigenic activities and explore these as the epigenetic drug-candidate targets in Group 3 (MYC-driven) MB. In this regard, we found that protein arginine demethylase Jumonji C domain-containing protein 6 (JMJD6), an emerging key epigenetic enzyme in cancers, is a novel regulator of MYC expression in MYC-driven MB. We observed high levels of JMJD6 that not only mirror MYC expression in the most aggressive MB but also correlate with poor outcomes in these patients. Knockdown of JMJD6 decreased MYC expression, cellular proliferation/survival and stemness in MYC-amplified MB cells. Mechanistically, our results revealed that JMJD6 forms complexes with proteins involved in maintaining and regulating the promoter-pause at super-enhancers, suggesting that JMJD6 can regulate MYC at the transcription level. Moreover, our in vivo analyses of JMJD6 inhibition, either with inducible gene knockdown or a pharmacologic small molecule inhibitor, demonstrated anti-MB potential with suppressed MYC expression. Based on this background and preliminary observations, we hypothesize that JMJD6 plays crucial roles in the most aggressive MB by regulating MYC expression and hence MYC-driven tumorigenesis. Accordingly, we hypothesize that targeting the JMJD6-MYC axis by JMJD6 inhibition can serve as a powerful therapeutic strategy for MYC-driven MB. Citation Format: Matthew Kling, Devendra Kumar, Sutapa Ray, Shantaram Joshi, Don Coulter, Nagendra K. Chaturvedi. JMJD6 as a novel tumorigenic factor and therapeutic target in group 3 (MYC-driven) medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3346.

Abstract 4397: Alpha-Ketoglutarate is a master regulator of epigenetic reprogramming in pancreatic ductal adenocarcinoma progression

Abstract Most cases of Pancreatic Ductal Adenocarcinoma (PDAC) are diagnosed after metastasis has occurred, making it crucial to find effective treatments for late-stage disease. Previously, our group found widespread epigenetic reprogramming during PDAC metastasis in the absence of additional driver mutations. These cells develop dependence on glucose metabolism through the oxidative pentose phosphate pathway (oxPPP). Inhibition of oxPPP with 6-aminonicotinamide (6AN) reverses the chromatin changes and restores cells to a less malignant state (McDonald et al. Nat Gen, 2017). To identify a clinically actionable mechanism for restoration, we hypothesized that oxPPP dependence leads to downstream metabolic changes that mediate chromatin state. Using untargeted metabolomic analysis, we found that alpha-ketoglutarate (AKG), a cofactor of lysine and histone demethylases, was downregulated by oxPPP inhibition (p=0.033) in a patient-derived xenograft lung metastasis cell line, A13Lg. Treatment of cells with 6AN and a cell permeable precursor of AKG largely reversed the chromatin changes seen on a western blot. Given that AKG is mainly produced by glutamine metabolism, we inhibited glutaminolysis with glutamine deprivation (Gln(-)) or the glutamine antagonist JHU083. Both treatments decreased AKG (Gln(-) p=0.002, JHU083 p=0.038) and prevented cell growth (Gln(-) p&amp;lt;0.0001, JHU083 p=0.0002). Glutamine-deprived cells showed decreased ability to form colonies (p=0.004), and JHU083 treated cells showed impaired migration in a Boyden chamber assay (p=0.003). Western blots showed a decrease in H3K27 acetylation, a marker of euchromatin, and an increase in H3K9 and H3K27 methylation, markers of heterochromatin, in response to both treatments. Further, homogenized time-resolved fluorescence (HTRF) assays on glutamine-deprived cells showed that H3K9 di- (p=0.0016,) and tri- (p=0.0003) methylation was increased. Adding cell permeable AKG to media without glutamine largely negated both the phenotypic and epigenetic changes. Using CUT&amp;RUN against H3K27Ac, we found that acetylation was depleted at promoters involved in tumor cell proliferation, invasion, and migration. Bulk RNA-sequencing also showed decreased expression of genes involved in these pathways. Epigenetic and phenotypic changes were not as apparent after treatment with CB839, a glutaminase 1 inhibitor, suggesting that broad inhibition of glutamine metabolism is necessary for chromatin restoration. Together, these data suggest that decreasing AKG in PDAC distant metastasis cells, through glutamine deprivation or JHU083, leads to chromatin changes that specify less malignant phenotypes, providing a rationale for targeting glutamine metabolism as a therapy for late-stage PDAC. *APF and MM are co-corresponding authors. Citation Format: Kenna Sherman, Jiaqi Cheng, Weiqiang Zhou, Hongkai Ji, Masahiro Maeda, Andrew P. Feinberg. Alpha-Ketoglutarate is a master regulator of epigenetic reprogramming in pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4397.

Abstract 7270: Combination targeting of SKP2 and KDM5B induces senescence in castration-resistant prostate cancer cells via AKT signaling

Abstract Prostate cancer (PCa) is the second-leading cause of cancer mortalities and morbidities in the United States and is the most commonly diagnosed malignancy in men. Despite the fact that androgen deprivation therapy is the first-line option to initial responses, most PCa patients invariably develop castration-resistant prostate cancer (CRPC). CRPC is a lethal type of malignancy, therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the combination treatment of small molecule inhibitors, SZL-P1-41, a SKP2 target and PBIT, a KDM5B target on PCa growth and progression; as well as to delineate the underlying mechanisms of suppressing CRPC. Literature reports that S-Phase Kinase Associated Protein 2 (SKP2) is upregulated in PCa and Lysine-specific demethylase 5B (KDM5B) serves as a histone demethylase with a crucial role in cancers. Studies showed that KDM5B is increased in human PCa and KDM5B knockout decreases carcinogenic properties of PCa cells. We previously reported that SKP2 loss partially decreases the growth of prostate tumors and that KDM5B levels are reversely regulated by SKP2 in PCa cells. However, the mechanisms of KDM5B and SKP2 interplay on PCa malignancy is unknown. We hypothesize that combination of PBIT and SZL-P1-41 treatment will enhance anti-cancer effects on PCa progression by inducing cellular senescence. Here, we showed that inhibition of KDM5B and SKP2 decreased the proliferation of PCa cells, and KDM5B KO cells were more vulnerable to SKP2 inhibition. More importantly, a combined inhibition of KDM5B and SKP2 significantly blocked malignant progression and migration of PCa cells. Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of cellular senescence, which was measured via Western blot analysis and senescence-associated β-galactosidase (SA-β-Gal) staining. Taken together, our results show that combined inhibition of KDM5B and SKP2 is more efficacious in inhibiting proliferation and migration in CRPC cells, and this regimen would be an ideal therapeutic approach for treating CRPC. Citation Format: LaKendria K. Brown, Thanigaivelan Kanagasabai, Guoliang Li, Sherly I. Celada, Zhenbang Chen. Combination targeting of SKP2 and KDM5B induces senescence in castration-resistant prostate cancer cells via AKT signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7270.

Abstract 2883: HDAC6 mediates the radioresistance of NSCLC through LSD1, independent of its own deacetylation activity

Abstract Resistance to radiation therapy significantly affects the prognosis of solid tumors. Histone deacetylase 6 (HDAC6) is a stress-responsive lysine deacetylase that has emerged as a promising target for cancer therapy, with numerous clinical trials investigating interventions to modulate its activity. While HDAC6 appears to be linked to responses to DNA-damaging therapeutics, the reported responses and underlying mechanisms remain intriguing and varied. In this study, we elucidate a novel mechanism of radioresistance mediated by HDAC6 in non-small cell lung cancer (NSCLC). We classified nine NSCLC cell lines into three groups based on their radioresistance using survival assays and correlated this resistance with the induced expression of six deacetylases. We conducted gain-of-function experiments (GOF) using plasmid transfection and loss-of-function experiments (LOF) employing inhibitors or RNAi. Furthermore, we quantified the repair of damaged dsDNA through FACS-based GFP assays and assessed the mechanism of homologous recombination repair (HRR) end-resection via single-strand quantitative PCR. Co-immunoprecipitation assays, cancer stem cell enrichment, DNA damage-induced senescence assays, reverse-phase protein assays (RPPA), and RNA sequencing were also performed to investigate gene expression changes resulting from the loss of HDAC6. Our findings revealed that HDAC6 induction by radiation is strongly correlated with NSCLC resistance to radiation leading to CSC survival and escape from radiation led senescence LOF of HDAC6 through RNAi or inhibitors suppressed resistance, while GOF achieved through wild-type or deacetylase activity-deficient mutant transfection, induced resistance. Importantly, this resistance mechanism was independent of deacetylation activity. RNAi-mediated loss of HDAC6 reduced both HRR and non-homologous end-joining, whereas pharmacological inhibition of HDAC6 activity did not. Notably, DNA end-resection, a critical step in HRR, was decreased by RNA interference but not affected by the inhibitors. RPPA assays demonstrated that Histone demethylase 1A (LSD1/KDM1A) levels decreased significantly upon HDAC6 RNA interference but remained unaffected by pharmacological intervention. Loss of LSD1 alone resulted in decreased HRR and end-resection, likely mediated through HDAC6-induced ubiquitination. Moreover, overexpression of LSD1 rescued the HDAC6 loss-induced sensitization of NSCLCs. In summary, our study unveils a novel mechanism by which HDAC6 mediates HRR after radiation through the stabilization of LSD1. This mechanism operates independently of deacetylase activity. Therefore, our findings suggest that interventions targeting both deacetylation and non-deacetylation roles of HDAC6 should be considered to overcome HDAC6-derived radioresistance in NSCLCs and enhances the radiation therapy efficacy. Citation Format: Sojung Ha, Hyejin Kim, Hani Lee, Seokgyeong Choi, Ho-young Lee, Woo-Young Kim. HDAC6 mediates the radioresistance of NSCLC through LSD1, independent of its own deacetylation activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2883.

Abstract 3237: KDM1A/LSD1 inhibition enhances chemotherapy response in ovarian cancer

Abstract Background: Ovarian cancer (OCa) is the deadliest of all gynecological cancers. The standard treatment for OCa is platinum-based chemotherapy, such as carboplatin or cisplatin in combination with paclitaxel. Most patients are initially responsive to these treatments; however, nearly 90% will develop recurrence and inevitably succumb to chemotherapy-resistant disease. Recent studies have revealed that the epigenetic modifier lysine-specific histone demethylase (KDM1A/LSD1) is highly overexpressed in OCa. However, whether KDM1A plays a role in chemoresistance and whether its inhibition enhances chemotherapy response in OCa is unknown. In this study, we explored the hypothesis that KDM1A inhibition will sensitize OCa cells to standard-of-care chemotherapy. Methods: Transduction of KDM1A-specific shRNA was used to generate KDM1A knockdown (KD) cells. Cell viability, colony formation, and apoptosis assays were used to determine the impact of KDM1A KD on sensitization of SKOV3, OVCAR8, and OVCAR5 OCa cells to cisplatin, carboplatin, and paclitaxel. The combination effect of KDM1A inhibitors (NCD38 and SP2509) and chemotherapy drugs was further determined. Mechanistic studies were conducted using RNA-sequencing, RT-qPCR, and Western blotting. In vivo efficacy ofKDM1A knockdown with or without chemotherapy was determined using OCa orthotopic xenograft models. Results: Analysis of TCGA datasets revealed that KDM1A expression is negatively correlated with OCa survival, and its expression is high in patients who do not respond to chemotherapy. Further, Western blotting results show that treatment with chemotherapy drugs cisplatin, carboplatin, and paclitaxel increased KDM1A expression in OCa cells. KDM1A-KD or KDM1A inhibition sensitized OCa cells to chemotherapy drugs in reducing cell viability, survival, and enhancing apoptosis. Moreover, knockdown of KDM1A sensitized carboplatin-resistantA2780-CP70 cells to carboplatin treatment. RNA-seq studies revealed that KDM1A-KD and cisplatin-regulated genes showed negative correction with epithelial-mesenchymal transition (EMT) and positive correlation with apoptosis-related pathways. Importantly, KDM1A KD in combination with cisplatin significantly reduced the tumor growth in the orthotopic OCa xenograft model. Conclusion: These results suggest that the combination of KDM1A inhibitors with chemotherapy is a viable therapeutic approach for the treatment of OCa Citation Format: Jessica D. Johnson, Yihong Chen, Sridharan Jayamohan, Yi He, Diksha Jamwal, Neetesh Pandey, Edward Kost, Ratna K. Vadlamudi, Gangadhara R. Sareddy. KDM1A/LSD1 inhibition enhances chemotherapy response in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3237.

Abstract 3011: The loss of an orphan nuclear receptor NR2E3 augments Wnt/β-Catenin signaling via epigenetic dysregulation that links to the Sp1-β catenin-p300 interactions in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) stands as the fourth leading cause of global cancer-related mortality due to late-stage detection, tumor heterogeneity, and drug resistance. The pivotal role of epigenetic factors in HCC development and progression necessitates the identification of critical epigenetic regulators. The orphan nuclear receptor NR2E3 (Nuclear receptor subfamily 2 group E, Member 3), primarily associated with retinal development, has emerged as a potential tumor suppressor. Earlier studies showed that the loss of NR2E3 leads to epigenetic repression of the estrogen receptor α (ER) through the involvement of Lysine Specific histone Demethylase 1 (LSD1). NR2E3 also forms a transcriptional complex with Sp1, governing the aryl hydrocarbon receptor (AHR) expression, a crucial factor in liver xenobiotic metabolism. In vivo, NR2E3 ablation results in p53 inactivation and severe liver injuries when exposed to liver toxicants, highlighting its role in activating p53. Nonetheless, the precise tumor suppressive and epigenetic role of NR2E3 in HCC remains unclear. HCC patients expressing low NR2E3 exhibit unfavorable clinical outcomes, aligning with heightened activation of the Wnt/β-catenin signaling pathway. The murine HCC models utilizing NR2E3 knockout mice show accelerated liver tumor formation and progression accompanied by enhanced activation of the Wnt/β-catenin signaling pathway and inactivation of the p53 signaling pathway. At a cellular level, losing NR2E3 increases the acquisition of aggressive cancer cell phenotype and tumorigenicity and upregulates vital genes in the Wnt/β-catenin pathway with enhanced chromatin accessibility. This event is mediated through the increased formation of active transcription complex involving Sp1, β-catenin, and p300, a histone acetyltransferase, on the promoters of target genes. Correspondingly, treatment of p300 inhibitor C646 decreased the expression of critical genes in the Wnt/β-catenin signaling pathway. These findings demonstrate that the loss of NR2E3 promotes Wnt/β-catenin signaling activation at cellular, organismal, and clinical levels. In summary, NR2E3 is a novel tumor suppressor that maintains epigenetic homeostasis, thereby preventing activation of Wnt/β-catenin signaling that promotes HCC formation. Citation Format: Yuet-Kin Leung, Sung-Gwon Lee, Jiang Wang, Shuk-Mei Ho, Nancy Rusch, Chungoo Park, Kyounghyun Kim. The loss of an orphan nuclear receptor NR2E3 augments Wnt/β-Catenin signaling via epigenetic dysregulation that links to the Sp1-β catenin-p300 interactions in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3011.

Abstract 7208: Targeting histone demethylase KDM6B to overcome cisplatin resistance in testicular germ cell tumors

Abstract Testicular germ cell tumors (TGCTs) represent the most common carcinoma of young men and are believed to arise from the transformation of primordial germ cells (PGCs). TGCTs are highly curable with platinum-based chemotherapeutics. However, resistance to cisplatin does occur, leading to therapeutic failure with very limited further therapy options. Previously we demonstrated polycomb and histone H3K27me3 methylation is repressed in cisplatin resistance TGCT cells. In TGCT patients the expression of H3K27 histone methylase EZH2 and H3K27 histone demethylase KDM6B is significantly associated with recurrence free survival. Induction of H3K27 methylation using demethylase inhibitor JSK-J4 or knockdown of KDM6B resulted in restored cisplatin sensitivity in cisplatin resistant TGCT cells. Furthermore, Inhibition of H3K27 demethylase by GSK-J4 or genetic knockdown of KDM6B sensitized TGCTs to cisplatin in vivo suggesting potential clinical utility of targeting H3K27me3 in refractory TGCTs. To explore underlying mechanisms H3K27me3 ChIP-seq was performed that demonstrated genome-wide loss of H3K27me3 in cisplatin resistant cells. Integrative analysis of H3K27me3 ChIP-Seq and transcriptomics is ongoing. Together findings suggests that repression of H3K27 methylation may be a viable approach to overcome treatment failure in cisplatin resistant TGCTs. Citation Format: Ratnakar Singh, Doha Shokry, Raya I. Boyd, Brayden C. Rennels, Christine Powell, Mehwish Kahn, Sarah J. Freemantle, Michael J. Spinella. Targeting histone demethylase KDM6B to overcome cisplatin resistance in testicular germ cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7208.